Identification of endophytic fungi with ACC deaminase-producing isolated from halophyte Kosteletzkya Virginica

ABSTRACT Seashore mallow (Kosteletzkya virginica), as a noninvasive perennial halophytic oilseed-producing dicot, is native from the Gulf to the Atlantic coasts of the U.S. The purpose of our research was to investigate 1-aminocyclopropane-1carboxylic acid deaminase (ACCD) producing endophytic fungi from K.virginica. A total of 59 endophytic fungal strains, isolated from roots in K.virginica of seedlings, were grouped into 12 genera including in Penicillium, Aspergillus, Fusarium, Trichoderma, Rhizopycnis sp., Ceriporia Donk, Trametes sp., Schizophyllum commune sp., Alternaria, Cladosporium, Cylindrocarpon, and Scytalidium according to sequences of ITS. The ACD activity of 10 endophytic fungi isolated was detected. T.asperellum had the highest ACC deaminase activity among all 10 isolated genera of fungal strains, followed by T. viride. Dry weight and fresh weight of plant, plant height, root length, SOD activity, and chlorophyll content of wheat and soybean inoculated with T.asperellum or T. viride was increased compared with non-inoculated control plants under non salt or salt stress. Further analysis showed that T.asperellum or T.viride strains induced downregulation of the expression of ethylene synthesis-related genes such as ACC oxidase (ACO) and ACC synthase (ACS), thereby reducing ethylene synthesis and damage to plants under salt stress. These endophytic fungi can be used as alternative bioinoculants to increase crop yield in saline soil.


Introduction
Soil salinity is frequently a limiting factor for cultivation of agricultural crops in arid and semiarid regions, which causes osmotic, ionic, and oxidative stress in plants and thus affects important morphological, physiological, and metabolic biological processes of plants, leading to serious loss of production every year. 1 According to the FAO, 20% of the world's irrigated and 2% of dry lands have been affected by salinity. 2 Halophyte plants have primary and secondary mechanisms to resist salt stress. The primary mechanism focuses on increasing the intracellular osmotic pressure to expel Na + from plant cells. 3 A secondary mechanism could be the endophytic association between plant and rhizobacteria named as plant growth-promoting rhizobacteria (PGPR), which are able to improve the plant growth in abiotic stress conditions. 3 PGPR is divided into three functional groups: plant growthpromoting bacteria (PGPB), biocontrol-PGPB, and plant stress homeo-regulating bacteria (PSHB). 4 In the symbiotic process, some endophytic bacteria promote growth of the plants by producing siderophores, solubilizing phosphorus, secreting auxin, 5 and synthesis of ACC deaminase (ACCD).
It was reported that when PGPR with ACCD was inoculated on the surface of rape seeds, they could absorb and utilize ACC exuded from seed coat during rape seed germination, and could effectively reduce ACC content in rape seeds and ethylene release, thus promoting root elongation and improving the salt tolerance of the seedling. 6,7 Likewise, some endophytic fungi were used to promote plant growth and resist the stress of drought and insect pests. Through symbiotic culture of Poplar with a variety of fungi, it was found that these symbiotic fungi could regulate its growth and development by promoting absorption of its nutrients, and have a positive effect on the prevention of plant diseases and insect pests, help resisting salt stress. 8 Arbuscular mycorrhizal fungi (AMF) promoted the growth and yield increase of strawberry, 9 and thus played an important role in the growth and development of the plant. 9,10 During this process, the endophytic fungi established a mutually beneficial relationship with the plants. Synthesizing ACCD was one of the important reasons for promoting plants growth. [11][12][13] During exposure to salt stress, plants resulted in stress hormone ethylene. ACCD is able to catalyze the conversion of ACC, the immediate precursor of ethylene synthesis in plants, to ammonia and α-ketobutyrate and thus promotes plant growth. 7,14 Previous studies showed that some endophytic bacteria and fungi producing ACCD not only reduced the level of ACC in plants, thereby reducing the ethylene level and mitigating biotic and abiotic stresses in plants, but also produced other biologically active substances to support the growth of their host. [15][16][17][18] Studies have shown that PGPR with ACCD activity could decompose ACC secreted by plant roots into α-butanone acid and ammonia, which could be used as carbon source and N source for self-utilization, respectively, thereby reducing ACC concentration in root system and generating ACC concentration difference between inside and outside of root cells, thus promoting ACC secretion from root system far from short to outside and thus reducing endogenous ethylene level in plants. Inoculation with this type of PGPR could significantly reduce the negative effects of salt stress and drought stress on the growth and yield of wheat, pea, rape, tomato, and others [ 19-22 23, 24 ] and help plants resist the damage of tissue hypoxia. 25,26 Therefore, PGPR with ACCD was used to minimize the impact of soil salinity on agriculture production. 27,28 Some halophytes establish and maintain effective associations with plant growth-promoting endophytic bacteria or fungi to help them grow well in saline soil. 29 Ethylene response factor (ERFS) is involved in abiotic and biotic stress processes, especially playing an important role under salt stress. 30 Similarly, EILs have conservated binding sequences of plantspecific transcription factors that participate in ethylene response. 31 In this study, endophytic fungi were isolated from seashore mallow (K.virginica) native in Atlantic coasts of the U.S. and their activities of ACCD were characterized. Then, the effects on wheat (Triticum aestivum) and soybean of these endophytic fungi were assessed. Expressions of key genes of plants, ACC synthase (ACS) and ACC oxidase (ACO), which plays an important role as the rate-limiting enzyme in ethylene synthesis induced by endophytic fungi containing the highest activities of ACCD were analyzed. And the expression levels of ERFs and EILs which play an indispensable role in the corresponding pathways of ethylene signaling were detected. Our results showed that the promotional effect of these fungi on plant growth is attributed to their regulation on pathways of ethylene synthesis, so these endophytic fungi could be used as potential bio-inoculants to sustain crop yield production under environmental stress conditions.

Isolation of endophytic fungi from roots of K. virginica seedlings
K.virginica seedlings were collected from plants which were grown in farmland alongside the Huanghe River in Zhengzhou City, Henan Province in China. To disinfect roots, they were rinsed with running water to get rid of the soil and dried in the air. Next, the roots of the samples were washed with distilled water and disinfected with 70% ethanol. Then, the roots were disinfected with 3% sodium hypochlorite. The root sections were cut into 5 mm long sections for experiments. Surface sterilization was verified by the last sterile distilled water after washing the samples, 100 µl of the final rinse water was plated on Potato Dextrose Agar (PDA) and incubated at 28°C. After thorough disinfection, hyphae or spores were removed with a dissection needle and inoculated on PDA with chloramphenicol, purified repeatedly, and the finally purified strain is inoculated on a slant culture medium for standby after being cultured at room temperature (25-30°C) for five days. The purified strain was inoculated onto a slope of inclined PDA medium for storage. The fungal colonies were selected according to the morphological and pigment production characteristics after incubation. 32

Morphological identification of endophytic fungi
The strains were inoculated on PDA and the growth and morphological characteristics were observed. Microscopic identification: Drop a drop of sterile water in the center of the slide, pick a small piece of hyphae into the water with the inoculation needle, add a cover glass, and observe with microscope. 33 Fourteen species of fungal strains colony screened were further purified on PDA medium plates and isolated single colony was transferred on slopes of PDA medium. All the isolates were preserved in a liquid medium with 50% glycerol at −80°C.

Identification of endophytic fungi
After the single colony of fungi is preliminary identified according to the standard microscopic morphology such as pattern of spore production, morphology of spores, color of aerial mycelium, texture and shape of strain colony, their exudates and growth rate. Isolates of fungi were incubated in PDA for 5 days at 28°C. The genomic DNA of isolates was extracted using CTAB method, Primer pairs ITS1(5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (3'-TCCTCCGC TTATTGATATGC-5') were used to amplify ribosomal internal transcribed spacer (ITS) of fungi. 34 The thermocycler program for clone of ITS sequence is as follows: 94°C for 2 min; 40 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 1 min, followed by a final extension of 72°C for 5 min. Agarose gel DNA purification kit (GE Healthcare, Buckinghamshire, UK) was used for purification of PCR products. Amplified DNA products were sequenced by Sangong (Shanghai, China). The DNA-ITS region sequence data were deposited separately to GenBank (http://blast. ncbi.nlm.nih.gov/blast.cgi) to blast their highly similar sequences.

Phylogenetic analyses
Multiple alignment searches were performed using the program CLUSTAL W. Phylogenetic analyses were performed by Neighbor-joining (NJ) method using MEGA 6.0. 35

ACC deaminase activity
Fungi with ACCD were screened using Penrose,s method. 6 Dissolved the α-ketobutyrate (Aladdin Industrial Corporation) into 0.1 M Tris-HCl pH 8.5 and made the stock solution. The solution is diluted with the same buffer to make a 0.5, 1.0, 1.5, 2.0, 2.5, 3 mmol·L −15 solution from which a standard concentration curve is generated. Separately, 200 μL lysate was mixed with 1.4 mL 0.56 M HCl, and 300 μL of 2,4-dinitrophenylhydrazine (Aladdin Industrial Corporation) reaction solution was added for the reaction at 30°C for 30 min, during which time the α-ketobutyrate is derivatized as a phenylhydrazone. The color of the phenylhydrazone is developed by the addition of 2.0 ml 2 M NaOH. The absorbance of the mixture was measured at 540 nm. One tube which was not added ACC was recorded as OD (fungi extract) , the tube of adding ACC was recorded as OD (fungi extract+ α −ketobutyrate) . OD( α-ketobutyrate) will be calculated by the formula: OD (α −ketobutyrate) = OD (fungi extract+ α −ketobutyrate) -OD (fungi extract) .

Measuring of physiological parameters of plants
T.asperellum and T.viride were grown on PDA liquid medium for 5 days. Centrifuge their fermentation to collect the hyphae and resuspend it in PBS to make hyphae suspension. The seeds of wheat (Yuguan 35) and soybean (Williams) were treated with 70% ethanol for 1 min and then with 5% sodium hypochlorite for 10 min. Then, the seeds were washed thoroughly in sterile water and cultivated with a temperature of 28/22°C and a photoperiod of 16 h/8 h (day/night) for germinating. The soybean and wheat seedlings which grow for 15 days were chosen and treated with resuspension of T. asperellum and T. viride for 5 days (5 ml/plant). Next, the soybean and wheat seedlings inoculated with T.asperellum or T.viride were cultivated for 15 days under the treatment of 0.5% NaCl. The experiment was conducted in the completely randomized design (CRD) with three replications. Samples were collected for analysis of phenotypic traits and activities of antioxidant enzymes. 36 The plant height, root length, dry weight, fresh weight, and chlorophyll content were measured according to Wen's method. 37 The plant leaves were washed and cut into pieces, and the leaves (0.1 g) were placed in each test tubes. Acetone and anhydrous ethanol (1:1) was added and treated dark overnight until the leaves were completely white. The absorbance was measured at 663 nm and 645 nm. Chlorophyll content will be calculated by the formula: Ca = V(12.7× OD 663 -2.69× OD 645 )/1000 w Cb = V(22.9× OD 645 -4.68× OD 663 )/1000 w C = Ca+Cb V: extraction volume; w: blade weight The SOD activity was detected with the Nitroblue tetrazolium photochemical reduction method, the fresh plant samples (0.5 g) were immediately smashed with liquid nitrogen. The samples were incubated in reaction buffer containing 5 mL of 50 mM phosphate buffer (pH 7.0), then were centrifuged for 20 min at 12000 g (4°C) . Next, the supernatant was gathered as a crude enzyme extract. The SOD activity was detected at 560 nm within 1 min.

Reverse Transcription Quantitative PCR (RT-qPCR) analysis of transcript abundance
RT-qPCR was performed to analyze the effects of T.asperellum and T.viride on Arabidopsis Thaliana under salt stress. First-strand cDNA synthesis was performed according to manufacturer's instructions (HiFi-MMLV cDNA kit CW0744s). Reverse transcriptase quantitative RCR was carried out using an Ultra SYBR Mixture ((LOW ROX) CW2601M) with Rotor-Gene (RG-3000). Actin was regarded as an internal control gene. The primer sequence is shown in Table 1.

Statistical analysis
The data were statistically analyzed using Graphpad Prism 8.0. The values are presented as mean ± standard deviation. P-values < 0.05 were considered significant and P-values < 0.01 were considered highly significant.

Diversity of isolated endophytic fungi from K. virginica
Some fungi isolated from K. virginica were identified ( Figure 1).  Table 1). The sequence data of ITS of isolated fungi was submitted to GeneBank under the accession number (OP117137~ OP458826). Molecular identification of part of the endophytic fungi from K.virginica was based on similarity analysis of ITS region (Figure 2). Different genera of endophytic fungi were identified based on ITS sequences (nuclear ribosome DNA internal transcribed spacer) (shown in Table 2).

Morphological characteristics of endophytic fungi
Due to the coevolutionary processes that occurred between plant roots and fungi, today a diverse array of fungi is able to penetrate root tissues and finally establish a symbiotic relationship with hosts. In order to understand the functions of endophytic fungi, morphology characters of their spores and mycelium of seven isolates were observed using microscope. On PDA medium plate, the diameter of the isolate of T. asperellum (LT8) (Figure 3a) reached 75-80 mm after five days at 25°C. At this time, it's color on the surface was white and it displayed as loose texture. After some time, the colony morphology of LT8 become flocculent, the color gradually changed into gray-green, while colorless at the bottom. Their conidia were oval, nearly colorless single cells. When gathered, they exhibited as yellow-green color, with smooth wall. The conidiospores looked like branch of the pine and cypress, which were divided into smaller alternate or opposite, bundle branches at the apex (Figure 3a). The colony of T. funiculosum (LT2) cultured on PDA medium displayed as velvety texture, with a diameter of 20-25 mm for five days. It then developed a dark green flocculent hyphae at the central part, accompanied with colorless exudate. The top of conidiophore gradually represented as branched structure that looked like rough broom. The shape of conidia was spherical, with nodular protrusions. The conidia chain displayed as dense and short columns (Figure 3b). The isolate (LT13) of C. lacerate displayed as white, thick and strong fluffy texture. The color of the colony is light yellow at the bottom of PDA medium. The hyphae were non-septum, tangled, most of which were produced on the ground (Figure 3c). The colony of A. flavus (LT3) grew rapidly on the medium plate. Its diameter reached 45-50 mm at 25°Cafter seven days. It displayed as loose and velvet-like texture, with indistinct radial furrows in the middle. The colony color was light green at the first stage, then grass-stained green on the surface, while slightly brown at the bottom. The conidiophore was colorless and rough. The conidia displayed a rough globular texture (Figure 3d). The colony of (LT6) F. oxysporum displayed as thick and flocculent texture, the hyphae were white. Color of colony was different, which was whitish, pale pink, or fleshcolored. Microconidia was oval unicellular, which clustered into pellets at the apex of conidiophore. Macroconidia took on shape of sickle. Chlamydospore produced at the apex or in the middle (Figure 3e). The colony of A. brassicae (LT10) grew sluggishly on the plate. The brown hyphae had substantial septate branches. The conidia spores displayed as blackish brown beaked structure, with trandiaphragm and mediastinum. There was rarely chlamydospores at the apex of dark-colored conidiophore ( Figure 3f). The colony of A. tenuissima. (LT11) on PDA plate reached 70-75 mm in diameter at 25°C for one week. The hyphae is divided into brown, septate branches. There were few hyphaes on the ground. The conidia were long oval-shaped, which arranged in chains (Figure 3g).  (Table 3).

Wheat seedlings growth inoculated with T. asperellum (Ta) and T.viride (Tv) under salt stress
To test the effect of endophytic fungi on the growth of plant, T. asperellum (Ta) and T.viride (Tv) with higher ACCD activity among all of isolated fungi was selected to inoculate the wheat seedlings. In normal conditions (without NaCl), the fresh weight, dry weight, root lengths, and plant height of wheat seedlings inoculated with T.viride increased by 5.93%, 3.32%, 11.01%, and 3.69%, respectively, compared with the control group. After inoculation with T.asperellum, in normal conditions (without NaCl), the fresh weight, dry weight, root lengths, and plant height, increased by 6.30%, 6.44%, 24.18%, and 5.89%, respectively. Under salt-stress conditions, the fresh weight, dry weight, root lengths, and plant height, inoculated with T.viride increased by significantly by 2.28%, 4.17%, 11.32%, and 4.78% compared with the control seedlings. Meanwhile, the fresh weight, dry weight, root length,   and plant height inoculated with T.asperellum under salt-stress condition increased significantly by 17.70%, 6.85%, 26.53%, and 5.76% Figure 4(a -d).
The chlorophyll content in salt stress conditions was lower than that in control conditions. However, the chlorophyll content of wheat inoculated with T.viride and T. asperellum increased by 32.88% and 26.52% compared with that of non-inoculated under salt-stress conditions, respectively (Figure 4e). These results indicated that T.viride and T.asperellum enhanced plant tolerance to salt stress by means of chlorophyll accumulation.
Our results clearly showed that the SOD activities in the plants were higher than the activities of all other treatments (including only salt, no salt, no T.viride and T.asperellum) under salt-stress conditions (Figure 4f). Therefore, these results indicated that T. viride and T.asperellum promoted plant tolerance to salt stress through the accumulation of antioxidant enzymes.
(Ta, Tv represents T.asperellum and T.viride respectively). The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P value < .05).

Effect of Inoculation with T.asperellum (Ta) and T. viride (Tv) on soybean seedlings growth under salt stress
The root of soybean seedlings was affected by T.asperellum and T.viride under 0.5% NaCl salt stress. In normal conditions (without NaCl), the dry weight, fresh weight, root lengths and plant height of soybean seedlings inoculated with T.viride increased by 9.26%, 0.86%, 4.67%, and 8.52%, respectively, compared with the control group. After inoculation with T. asperellum, in normal conditions (without NaCl), the dry weight, fresh weight, root lengths and plant height, increased by 3.79%, 5.89%, 20.33%, and 4.35%, respectively. Under saltstress conditions, the fresh weight, dry weight, root lengths, and plant height, inoculated with T.viride increased by significantly by 17.16%, 17.04%, 4.17%, and 14.80% compared with the control seedlings. Meanwhile, the fresh weight, dry weight, root length, and plant height inoculated with T.asperellum under salt-stress condition increased significantly by 14.56%, 26.40%, 17.97%, and 2.83% (Figure 5a-d). The results showed that T. asperellum and T. viride could significantly increase the growth of soybean seedlings under salt stress. All of them can improve the activity of SOD enzyme of soybean seedlings, enhance the oxygen-free radical scavenging system, reduce the damage of salt stress on soybean seedlings, improve the growth of soybean plants under salt stress, and enhance the tolerance of soybean to salt and the resistance of plants to abiotic stress.
(Ta, Tv represents T.asperellum and T.viride respectively) The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P value < .05).

Effect of T. asperellum and T.viride on the expression of ethylene synthesis-related genes of A. Thaliana under salt stress
In the ethylene biosynthesis in plants, methionine is the biosynthetic precursor of ethylene. Firstly, S-adenosyl methionine (SAM) is synthesized by converting methionine to s-adenosyl l-methionine. Then, ACC synthase (ACS), the pyridoxal enzyme, converts SAM into 1-Aminocyclopropane-1-carboxylate (ACC) and methyl-thioadenosine. ACC oxidase (ACO) finally catalyzes the conversion of ACC to ethylene, HCN, CO2, and water. 38 A burst of reactive oxygen species (ROS) is observed within minutes after the onset of salt stress, which activates multiple signaling cascades of downstream 39

Discussion
Salinity stress has devastating effects on plant growth and reproduction, resulting in reduced yield. 39 As part of the symbiosis, the plant provides certain nutrition and space of reproduction and distribution of PGPR. 40 PGPR strains are capable of promoting plant growth by suppressing phytopathogens and alleviating abiotic stress to promote plant growth. 41 Some PGPR with ACCD producing promoted grow of plants under salinity stress due to lowering ethylene accumulation of their hosts. 42 In our experiment, 59 strains of fungi were isolated, and 12 genera were identified; 43 strains of bacteria were isolated from K. virginica, and 10 genera of bacteria were identified (data unpublished). The number of genera of fungi coexisting into K. virginica of roots in seedlings was more than that of bacteria. The majority of endophytic fungi were confirmed to be tolerant to salt stress. Among the fungal strains identified, T.asperellum, T.viride, P. funiculosum, A. ochraceus and F. oxysporum were the dominant species with salt tolerance. Few of genera of fungi such as Schizophyllum commune and Cladosporium sp. were not sensitive to NaCl (data unpublished). These salt tolerant endophytic may have contributed to the growth of K. virginica in saline soil.
Furthermore, 10 fungal isolates, among 14 isolates from K. virginica were screened out and detected their ACCD activity. A. flavus generally causes significant yield losses in cereal crops due to its mycotoxin producer. However, it is also used as a beneficial fungal to promote the development of plants. 43 F. oxysporum has been reported for plants growth promotion and IAA production. 44 Previous reports have documented that Penicillium and Trichoderma possess ACCD activity, 45,46 which was consistent with our results. ACCD hydrolyzes ACC (precursor of ethylene biosynthesis in higher plants) into alkali and α-ketobutyrate for use as a nitrogen source 28 and enhances plant growth under saline conditions [ 47 . 48 ]. Likewise, ACCD can protect plants from pathogenic microorganisms and drought stress. Trichoderma has been shown to have promoting effect on plant growth. 49 For example, T. asperellum isolated in plants grown along the marshy lands near Tarbela lake enhanced wheat tolerance under waterlogging stress. 50 This was consistent with the fact that the, T. asperellum and T.viride obtained from K. virginica had ACCD in our experiment. P. aurantiogriseum has been  reported to synthesize paclitaxel, which would facilitate metabolic engineering for the industrial production of paclitaxel from fungi. 51 A. ochraceus has phosphate-solubilizing properties and disease resistance to plants. Maybe ACCD of P. aurantiogriseum and A. ochraceus derived from plants help their hosts growing in saline soils or soils in arid and semiarid regions. The photoassimilates facilitate growth, maintenance, and osmotic adjustment during physiological processes and sink organs in plants. 52 Our research indicated inoculation with T.asperellum or T. viride increased chlorophyll of dicotyledonous soybean and monocotyledonous wheat under salt stress, implying that the two fungal isolates could counteract the suppression of photosynthesis under salt stress conditions. Salt stress disturbs the balance of active oxygen metabolism system in plants, resulting in the production of reactive oxygen species (ROS), such as the superoxide radical(O 2 − ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical (OH − ), which induces membrane lipid peroxidation in plant tissues, resulting in the destruction of a series of biochemical processes and subsequently reduced plant growth. 53,54 ROS can have adverse effect on growth and metabolism of plants. SOD was involved in scavenging ROS. Our results clearly showed that the SOD activities in the inoculated plants under salt stress conditions are higher than that of non-inoculated plants.
Earlier reports have demonstrated the role of PGPR in ameliorating salt stress through modulating the ethylene level. 12,55 Ethylene is perceived by five receptors ETHYLENE RESPONSE1 (ETR1), ETR2, ETHYLENE INSENSITIVE4 (EIN4), ETHYLENE RESPONSE SENSOR1 (ERS1). 56,57 ETR1 and ETR2 mediate the salt stress response through ABA and Ca2 + pathways. In the presence of ethylene, the C-terminal of EIN2 is cut and transferred to the nucleus, where it participates in the stabilization and accumulation of EIN3/ethylene insensitive 3-like1 (EIL1), thus inducing ethylene-responsive genes. [58][59][60] The ERF/EIL transcription factor, which are widely involved in the response of plants to various stresses, participated in ethylene signal pathway by binding downstream gene promoters to initiate their gene expression. 57,61 EIL proteins are the key components of ethylene signal transduction, 31 which play an important role in plant response to abiotic stress. A clear coexpression was observed between ERF6 and ERF11 under numerous biotic and abiotic stress conditions. 62 In our experimental records, the expression level of AT4G17490 (ERF6) inoculated with T. viride was remarkably decreased compared with that without inoculation under salt stress. The expression levels of AT1G50640(ERF3), and AT5G44210 (ERF9) were significantly increased under both biotic and abiotic stresses. 63 Expression of AT1G50640 (ERF53) and AT4G28140 (ERF54) have been induced in plant responses to cold and heat stress as well as lateral root development. 64,65 Here, they were significantly reduced after inoculation with T. viride and T. asperellum under salt stress in our experiment. ERF and EIL genes were down regulated in Arabidopsis seedlings inoculated with T.asperellum or T. viride. These results indicated inoculation with T.asperellum and T. viride decreased ethylene synthesis, thus improved the tolerance of seedlings to salt stress.
In the process of ethylene biosynthesis, ACO and ACS are important rate-limiting enzymes AtERF11 can interact with the ACS2/5 gene promoter to inhibit the expression of ACS2 and ACS5 genes, thereby negatively regulating the synthesis of ethylene. The regulation of ACS activity and ACC content in the cellular pathway ultimately determines the intracellular ethylene synthesis rate. 31,66 Previous studies have also demonstrated that under high salinity, ethylene level increased due to enhanced ACO activity. 67,68 After inoculation with T.asperellum, expression of ACO gene was reduced, which could be due to decrease in ACC content caused by activities of ACCD in the fungal isolates.
In conclusion, this study reported firstly on isolation and characterization of endophytic fungi of roots in K. virginica seedlings. Some fungal isolates with ACCD activity could be used to enhance salt tolerance of glycophytes in saline soil.